Vertebrate Biotechnology

Question 1

What is pronuclear injection and what are some advantages?

Answer 1

The simplest transgenic method

Was discovered in the 1980s

Widely applicable across species for adding genes in random locations

Lead to the development of other methods

Is the standard method for adding genes

Cheap, effective, proven, useful and can be used cross species

Question 2

Describe how pronuclear injection works

Answer 2

A small suction pipette holds a newly fertilised mouse egg in place using negative pressure

A glass pipette injects picolitres of DNA containing the DNA fragments of the transgene penetrates the egg cell membrane then the haploid nuclei from the egg and sperm which haven’t combined into a diploid yet

They are then grown in culture to a morula

The morula is then implanted into pseudo-pregnant females (have been mated with sterile males and have the needed hormonal changes)

The pups are then tested for presence of the transgene and then are bred with heterozygotes to produce a homozygous mouse

They are then bred to multiple generations to ensure successful uptake of the transgene in the germ cells and for any adverse side effects

Question 3

Why are mouse eggs used?

Answer 3

The standard model for transgenics

Breed rapidly

Have short generations

Are small

Give birth to 6-8 pups

Question 4

What DNA is injected in pronuclear injection?

Answer 4

A promoter which has been chosen according to the regulations of the application e.g. constitutive promoters have strong, constant expression whereas tissue specific promoters are regulated and their activity can be precisely controlled

An intron which contains the binding sites for transcription factors

An open reading frame containing the cDNA or DNA sequence

A polyadenylation site which allows the polyA tail to be attached to mRNA and is needed for the export of mRNA from the nucleus and translation

Question 5

What form should the transgene be in before injection?

Answer 5

Linear as they integrate into the genome at a higher frequency than circular plasmids so circular plasmids are cut using a restriction enzyme

Multiple linear copies are injected into the egg

Question 6

What are some problems with pronuclear injection?

Answer 6

Can only be used to add genes not modify or take away

Ectopic insertions as there is no control over where in the chromosome it is inserted

Multiple copies can join together in chains

Position effects of flanking chromosomal sequences can lead to silencing or enhancement effects thus the expression of the transgene will vary depending upon its position in the genome

The transgene is not always consistently inherited and could disrupt other genes if inserted incorrectly i.e. in a regulatory sequence for another gene

Question 7

What is homologous recombination?

Answer 7

Developed in the late 1980’s

Enables the precise modification of specific genes in embryonic stem cells

A single copy of the modified gene is modified in its normal chromosomal environment

Relies on the natural process of homologous recombination (exchange of DNA segment between DNA strands in chromosomes)

Applicable only to mice

Question 8

What are embryonic stem cells and what are the advantages of using them?

Answer 8

Also called a blastocyst and contains ~150 cells and are pluripotent

Are formed shortly after fertilisation thus are developmentally flexible

Embryonic stem cells from the blastocyst inner mass are cut out and grown in culture

Are capable of dividing into any cell type

Have a higher rate of homologous recombination than other cells

Question 9

Describe HR knockins

Answer 9

HR allows a transgene to be inserted into a precise position in a chromosome

Question 10

Describe HR knockouts

Answer 10

Insert and expression of a marker gene in a position that results in a natural gene being inactivated

An example is the introduction of neoR marker gene which makes cells survive exposure to neomycin thus positive selection (this is rare as it is more likely to randomly insert)

A second marker such as thymidine kinase is incorporated which kills the cells thus negative selection

Question 11

Describe how a knockout mouse is made

Answer 11

A mouse blastocyst has the embryonic stem cells removed from its inner mass and the targeting vector is introduced by electroporation (which opens the membrane channels) or lipofection (transgene forms complex with lipid and gently enters cells)

Positive/negative selection screens for embryonic stem cells with the targeting vector

The targeted embryonic stem cells are injected into blastocysts and mixed with natural embryonic stem cells (the blastocyst comes from a mouse with different fur colour than the original blastocyst) and is then injected into foster mothers

This results in the birth of chimeric mice with the transgene (have the cells of 2 genomes)

A normal mouse is mated with a chimeric mouse to produce desired heterozygous progeny

Further cross breeding is required for homozygous knockouts as only one allele on one chromosome has been modified

Question 12

Why is it not possible to grow a whole mouse from embryonic stem cells?

Answer 12

The embryonic stem cells are pluripotent not totipotent and as such are too developed

Need to return modified stem cells to a blastocyst and hope some of them differentiate into germ cells to the modification is inherited in future generations

Question 13

Why are knockouts important?

Answer 13

Allows for the studying of gene function and the mechanisms of diseases

Question 14

Describe how Dolly the sheep was made

Answer 14

Donor cells were removed from the mammary gland (fully differentiated cells) of a Finn Dorset Ewe (white face) and starved in a low-nutrient culture medium to arrest the growth cycle

An unfertilised egg cell is donated by a Scottish Blackface Ewe and has the nucleus removed to produce and enucleated egg cell

The donor cells and the enucleated egg cell are fused with electrical pulses and will behave as if it is a newly fertilised egg

The fused cell undergoes cell division and forms and embryo which is then implanted into a Scottish Blackface Ewe surrogate mother

Question 15

Describe how Dolly the sheep was made

Answer 15

Nuclear transfer

Took 177 attemps

Donor cells were removed from the mammary gland (fully differentiated cells) of a Finn Dorset Ewe (white face) and starved in a low-nutrient culture medium to arrest the growth cycle

An unfertilised egg cell is donated by a Scottish Blackface Ewe and has the nucleus removed to produce and enucleated egg cell

The donor cells and the enucleated egg cell are fused with electrical pulses and will behave as if it is a newly fertilised egg

The fused cell undergoes cell division and forms and embryo which is then implanted into a Scottish Blackface Ewe surrogate mother

Dolly was born with a white face thus taking on the phenotype of the donor cell not the egg donor or surrogate mother phenotype

Question 16

Why is the rat an important model animal?

Answer 16

Was the first mammal bred for research

Is the most widely used mammalian biomedical model and is the standard model for drug testing in clinical trials of human drugs and physiology whereas mice are used for genetics

Are larger than mice which makes the measurement of organs simpler

However rat embryonic stem cells are hard to culture so HR cannot happen

Question 17

What are ZFNs?

Answer 17

Zinc finger nucleases are hybrid fusion proteins which are used to cut DNA

FokI nuclease cleavage domain is combined with several zinc finger domains which have been engineered to bind to a specific target sequence

Question 18

How do ZFNs work?

Answer 18

FokI cleavage domain requires dimerisation to break the double strand and 2 ZFNs are designed to target sequences on each strand separated by a 6 bp cleavage site

The double strand break activates the DNA repair response and in 70% of cases, non homologous end joining (NHEJ) will occur involving short insertions or deletions to disrupt genes (knockout) and in the other 30% of cases homologous recombination (HR) will occur if a template sequence is introduced (knockin)

Question 19

Describe the research conducted in the first ZFN transgenic rat paper

Answer 19

Knockout rats were produced via embryo microinjection of ZFNs that targeted immunoglobin heavy chain μ locus (IgM) which is needed for B lymphocyte survival

Used a promoter that was only activated briefly in the early embryo (CAG promoter)

The vector was not incorporated into the genome but instead injected into the egg as a circular plasmid

Question 20

Describe how TALENs was used in cattle gene editing tests

Answer 20

Knockout of the myostatin gene (inhibits muscle development) in cattle and sheep using TALENs (engineered restriction enzymes with DNA binding and cleavage domains similar to ZFNs) injected into the egg cytoplasm which caused muscular hypertrophy in knockouts

Question 21

What are some unintended effects of CRISPR/Cas9?

Answer 21

Off target effects where there are unintended DNA breaks in regions with a similar sequence to the target site (this can be controlled using bioinformatic software of using modified Cas enzymes)

Mosaicism and multiple alleles where the editing persists beyond the one-cell stage and multiple alleles are created (extensive breeding and genotyping is needed to isolate the animals with the desired alleles)

Integration of the template vector when creating knockins

Question 22

Why were transgenic fish produced?

Answer 22

Increasing world demand for fish

Little selective breeding had been done in fish

Fish populations are decreasing

The potential for aquaculture (fish populations bred in pens off the coast for commercial production)

Question 23

How were the first transgenic fish made?

Answer 23

In 1985, goldfish expressing human growth hormone were made via direct injection of DNA into fertilised egg cytoplasm as the pronucleus was too hard to see

Question 24

How were AquAdvantgage salmon made?

Answer 24

Ocean pout antifreeze protein gene (AFP) 5’ promoter and 3’ terminator was linked to salmon growth hormone

The promoter allowed the growth hormone to be produced outside the pituitary gland and during all seasons

The growth rate not the final size was affected so transgenic salmon reached market size 1 year and required less food

Was under development for 30 years and had a long wait for approval from FDA due to non-existent regulations and pressure groups

Question 25

What were some of the sustainability claims made by AquaBounty?

Answer 25

The salmon ate 25% less than conventional farmed fish and had a high feed conversion compared to traditional farm animals

Inland production facility in Indiana prevented escape and mating with wild-type salmon which would cause declines in the natural population

Salmon were made sterile to prevent affecting natural populations in case of escape

The inland facility also allowed recycling and treatment of the water to prevent diseases, parasites and pollution

The fish were produced closer to the consumer than wild or farmed fish so there were reduced carbon dioxide emissions

Question 26

Describe how xenotransplantation works

Answer 26

Lack of supply from human donors means that animal organs can be made suitable for implantation into human hosts

Pigs are used as they are efficient breeders, have similar organ size to humans and have compatible life expectancies

Genes involved in rejection can be manipulated as the human immune system would recognise the organ as foreign

Question 27

Describe how gene knockout in pigs by nuclear transfer can aid xenotransplantation

Answer 27

Alpha 1,3 galactosyl transferase is not present in humans but adds a carbohydrate antigen to pig cells thus would be attacked by human immune system

Knockout in pigs by HR and nuclear transfer using cultured foetal fibroblasts were used

Question 28

Describe the use of human embryonic stem cells in therapeutic cloning

Answer 28

Human embryonic stem cells were isolated in 1988

Nuclear transfer of somatic cell into a enucleated donor oocyte then grow ESC in culture

Could grown patient’s own cells (to replace diseased cells/tissues) or correct genetic defects

Are immunologically compatible for transplant

Question 29

What are iPS cells?

Answer 29

Induced pluripotent stem cells

Reverse the differentiation by administering 4 transcription factors in viral vectors

Avoids the need to clone and kill human embryos to collect ESC

Question 30

How are iPS made?

Answer 30

Skin cells are collected

Using Oct4, Sox2, Klf4 and c-Myc viruses to reprogram the skin cells into ESC like iPS cells

This produces genetically identical iPS cells that will need to have mutations corrected and differentiated

They can then be transplanted into sick individuals

E.g. differentiate into blood stem cells and inject into patient with sickle cell anaemia